This invention relates generally to medical diagnostics for quantitative analysis of low density lipoprotein fractions and assignment of cardiac disease risk.
Lipoproteins
Lipoproteins are complex molecules found in the blood. One of the biological actions of lipoproteins in the circulatory system is to transport cholesterol for cellular use. Lipoproteins are subdivided into classes and subclasses on the basis of density, which is determined by equilibrium density ultracentrifugation and analytic ultracentrifugation, or size, which is determined by gel electrophoresis. The classes which have been assigned to various lipoprotein fractions are: very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL).
Ultracentrifugally isolated lipoproteins, analyzed for flotation properties by analytic ultracentrifugation in two different salt density backgrounds, allows for the determination of hydrated LDL particle density. (Lindgren et al, Wiley, 1992 p. 181-274.) A comparison of LDL particle densities from 642 subjects shows a distinct bimodality separating groups with particles greater than and less than 1.032 gm/ml. In the group with LDL, d..gtoreq.1.032 gm/ml, there is a higher correlation of coronary artery disease risk factors including elevated plasma triglycerides, increased body mass index and history of familial coronary disease.
The LDL class can be further divided into seven subclasses based on density or particle diameter using a preparative separation technique, equilibrium density gradient ultracentrifugation (EDGU). Elevated levels of specific LDL subclasses, LDL-IIIa, IIIb and IV a, and their related cholesterols, is the clinical finding which correlates most closely with increased risk for coronary artery disease, including atherosclerosis.
The determination of total serum cholesterol levels is used routinely as a diagnostic test. Because it is expensive and time-consuming, determination of the more predictive lipoprotein class and subclass distribution is typically ordered by physicians only for a very limited number of patients.
LDL Quantitation
Currently, the most widely used method for quantitating LDL cholesterol is the indirect Friedewald method (Friedewald, et al., Clin. Chem. Vol. 18, pp.499-502, 1972). The Friedewald assay method requires three steps: 1) determination of plasma triglyceride (TG) and total cholesterol (TC); 2) precipitation of VLDL and LDL, and 3) quantitation of HDL cholesterol (HDL-C). Using an estimate for VLDL-C as one-fifth of plasma triglycerides (TG/5), the LDL cholesterol concentration (LDL-C) is calculated by the formula: LDL-C=TC-(HDL-C+VLDL-C).
While generally useful, the Friedewald method is limited in its accuracy in specific cases. Errors can occur in any of the three steps, in part because this method requires that different procedures be used in each step. The Friedewald method presumes that VLDL-C concentration is one-fifth that of plasma triglycerides. When the VLDL of some patients deviates from this ratio, further inaccuracies occur. For patients with TG over 400 mg/dL, the Friedewald method cannot be used.
Otvos teaches a procedure for determining concentrations of lipoprotein subclasses which allows greater accuracy than Friedewald (U.S. Pat. No. 5,343,389, issued Aug. 30, 1994). Otvos initially obtains the NMR chemical shift spectrum of a blood plasma or serum sample. The observed spectrum of the whole plasma sample is then matched with weighted sums of NMR spectra of lipoprotein subclasses, which are stored in a computer software program. The weighting factors which give the best fit between the sample spectrum and the calculated spectrum are then used to estimate the concentrations of constituent lipoprotein subclasses in the blood sample. This procedure has the additional advantage of being rapid.
Another lipoprotein subfraction determination method that is used clinically is the Vertical Auto Profile (VAP), (Kulkarni, et al., J. Lip. Res. Vol. 35, pp.159-168, 1994). VAP uses a flow analyzer for the enzymatic analysis of cholesterol in lipoprotein classes separated by a short spin single vertical ultracentrifuge, and subsequent spectrophotometry and software analysis of the resulting data. While a useful advancement, this technique does not resolve the LDL into all seven subspecies isolated by ultracentrifugation.
Gel Separation
The subject inventors previously have developed a procedure using gradient gel electrophoresis for separation of LDL subclasses. The LDL subfractions separated by the gradient gel electrophoresis are comparable to those obtained by ultracentrifuge. This method makes a finer resolution of LDL subclasses available to research laboratories. It would be useful if gradient gel electrophoresis separation could be shortened and simplified so that it could be used in clinical laboratories as part of a routine screening of blood samples, to assign a risk factor for coronary artery disease.
A high resolution assay for measuring LDL cholesterol that would be accurate, direct, and complete, quantitating all major subclasses of LDL would be an important innovation in this technology. If inexpensive and convenient, it could be employed in a clinical laboratory setting. Ideally, clinicians could assign a patient risk factor for CAD, and correlate concentrations of LDL subclasses with the more commonly used LDL-cholesterol concentrations for conventional use.